This invention relates to modified hydrophobic membranes having at least a portion of their surface coated with a first molecule bound to a labeled second molecule for the first molecule and to analytical processes utilizing the modified hydrophobic membranes.
Molecules including biomolecules, such as proteins and nucleic acids, are applied to microporous membranes to facilitate detection of specific molecules within complex mixtures. The simplest detection system consists of a first molecule bound to the membrane and a second molecule used for the detection. The first molecule is derived from a sample, adsorbed on the membrane, and then reacted with the second molecule. The second molecules most commonly used for detection of proteins and nucleic acid first molecules are themselves labeled proteins, antibodies, lectins, or nucleic acids such as DNA or RNA. Application of the first molecule to the membrane relies on the membrane's inherent ability to adsorb protein and nucleic acids. To take full advantage of the membrane's adsorption capacity, hydrophilic membranes composed of polymers such as nitrocellulose or nylon are wet in aqueous buffer. Hydrophobic membranes such as those composed of polyvinylidene fluoride (PVDF) are wet first in alcohol, equilibrated in deionized water, and then equilibrated in aqueous buffer as disclosed, for example by Reig et al, "Applied and Theoretical Electrophoresis" 1988, Vol. 1, pgs. 59-60. The first molecule then is applied to the membrane directly or transferred from a gel by passive diffusion or in an electrical field. Although the detection protocol can be initiated immediately using the wet membrane, the membrane is often allowed to dry. The membrane must then be rewet before continuing.
In most cases, the amount of first molecule applied the membrane does not saturate the surface of the membrane. Thus, exposed polymer surface area at and surrounding the point of application provides sites for non-specific binding of the second molecule. To circumvent this problem, the first step in detection protocols involves incubation of the membrane in aqueous buffer containing compounds that block the exposed polymer surface area. Common blocking agents are bovine serum albumin (BSA), non-fat dry milk, gelatin, calf thymus DNA, salmon sperm DNA, and yeast tRNA.
Specific detection of first molecules on microporous membranes requires blocking of exposed binding sites prior to introduction of the second molecule as detectors. If the second molecule binds to exposed binding sites on the membrane, it will be unable to bind to the first molecule. Non-specific binding of the second molecule also causes higher background rendering it difficult to distinguish the signal.
Although efficient blocking of the membrane enhances the signal-to-noise ratio, there are several inherent problems with this step. First, depending on the blocking agent used and the first molecule being detected, the blocking step requires between 1 and 18 hours. Second, attachment of blocking agents to exposed areas of the membrane is not permanent. Proteins and nucleic acids adsorb to membranes through electrostatic, ionic or hydrophobic interactions. Since these interactions are noncovalent, blocking agents may desorb from the membrane after initial binding. Thus, blocking agents are included in vast excess in solutions containing the second molecule detection probes. In a similar fashion, first molecules may desorb from the membrane during incubation. Third, if the blocking agent has a higher affinity for the membrane than the first molecule, the first molecule can be displaced from the membrane, decreasing the signal intensity. Fourth, the blocking agent may non-specifically bind to the first molecule, preventing it from interaction with the second molecule detection probe. Fifth, the second molecule may non-specifically bind to the blocking agent either in solution or on the membrane. Non-specific interaction of the second molecule with the blocking agent is often corrected by the addition of detergents to the second molecule-containing solutions. Detergents, however, enhance the undesirable desorption of the first molecules from the membrane thereby reducing signal intensity. These difficulties require that blocking agents be screened for compatibility with the first and second molecules and that the incubation protocols be optimized to obtain a maximum signal.
Prior to the present invention, it was thought the membrane pore structure must be made accessible to aqueous solutions by a pre-wetting step, such as with methanol followed by exchange with water, so that molecules in aqueous diluent could properly interact with membrane-bound first molecules. This perceived requirement, in turn, promulgated the use of a blocking agent to be applied to the membrane over a suitable period of time followed by one or more suitable molecules the last of which comprises the second molecule which is used for detection. The intimate contact of second molecules with the membrane, in turn, necessitates time consuming washing steps to assure complete removal of unbound second molecules from the membrane surface and from the interior volume of the membrane which can lead to nonspecific signal or background. The time required to effectively wash the membrane extends the required analysis time and also represents a period during which the bound second molecule may desorb from the first molecule, thereby reducing the signal intensity.
Accordingly, it would be desirable to provide a hydrophobic membrane, having bound first molecules, which eliminates the need for a blocking agent and minimizes non-specific adsorption of second molecules utilized for detection. In addition, it would be desirable to provide a hydrophobic membrane having bound first molecules which, in turn, are bound to a labeled second molecule in the absence of a blocking agent. Such membranes would eliminate the need for a blocking agent and therefore would eliminate the need of a membrane wetting step to effect deposition of the blocking agent from aqueous solution.